Fluorescent Chromophore Research Articles

Planarity is one of the essential requirements for fluorescence in Red Fluorescent Protein chromophore analogs

Synthesis and photophysical properties of four red fluorescent protein (RFP) chromophore analogs are reported. The analogs in crystals are held by different non-covalent intermolecular interactions such as C–H....O hydrogen bonding, C–H....π, π....π, and halogen bonding interactions, stabilizing the molecular stacks. The carbonyl group in the imidazol-5(4H)-one ring generates diverse packing systems in the compound via intermolecular C–H....O hydrogen bonds. Compounds 1 and 2 adopt a non-planar geometry, while 3 and 4 are slightly bent. Compound 3 shows a donor-acceptor type of π-π interactions resulting in a lateral offset stacking that is stabilized by strong π-π stacking between the imidazolin-5-one ring and methoxy substituted benzene, along with halogen....halogen bonding interactions. Compound 4 displays a strong π-π stacking between two imidazol-5(4H)-one rings. The Hirshfeld surfaces of two-dimensional fingerprint plots were used to analyze the strength of intermolecular interactions in the structures. The photoluminescence is also tuned in these molecules by appropriate substitution. Thus, the azine compounds are completely non-fluorescent compared to the other two.

Observation of Cation Chromophore Photoisomerization of a Fluorescent Protein Using Millisecond Synchrotron Serial Crystallography and Infrared Vibrational and Visible Spectroscopy.

The chromophores of reversibly switchable fluorescent proteins (rsFPs) undergo photoisomerization of both the trans and cis forms. Concurrent with cis/trans photoisomerisation, rsFPs typically become protonated on the phenolic oxygen resulting in a blue shift of the absorption. A synthetic rsFP referred to as rsEospa, derived from EosFP family, displays the same spectroscopic behavior as the GFP-like rsFP Dronpa at pH 8.4 and involves the photoconversion between nonfluorescent neutral and fluorescent anionic chromophore states. Millisecond time-resolved synchrotron serial crystallography of rsEospa at pH 8.4 shows that photoisomerization is accompanied by rearrangements of the same three residues as seen in Dronpa. However, at pH 5.5 we observe that the OFF state is identified as the cationic chromophore with additional protonation of the imidazolinone nitrogen which is concurrent with a newly formed hydrogen bond with the Glu212 carboxylate side chain. FTIR spectroscopy resolves the characteristic up-shifted carbonyl stretching frequency at 1713 cm-1 for the cationic species. Electronic spectroscopy furthermore distinguishes the cationic absorption band at 397 nm from the neutral species at pH 8.4 seen at 387 nm. The observation of photoisomerization of the cationic chromophore state demonstrates the conical intersection for the electronic configuration, where previously fluorescence was proposed to be the main decay route for states containing imidazolinone nitrogen protonation. We present the full time-resolved room-temperature X-ray crystallographic, FTIR, and UV/vis assignment and photoconversion modeling of rsEospa.

Heavy-atom engineering of thermally activated delayed fluorophores for high-performance X-ray imaging scintillators

The architectural design and fabrication of low-cost and reliable organic X-ray imaging scintillators with high light yield, ultralow detection limits and excellent imaging resolution is becoming one of the most attractive research directions for chemists, materials scientists, physicists and engineers due to the devices’ promising scientific and applied technological implications. However, the optimal balance among X-ray absorption capability, exciton utilization efficiency and photoluminescence quantum yield of organic scintillation materials is extremely difficult to achieve because of several competitive non-radiative processes, including intersystem crossing and internal conversion. Here we introduced heavy atoms (Cl, Br and I) into thermally activated delayed fluorescence (TADF) chromophores to significantly increase their X-ray absorption cross-section and maintaining their unique TADF properties and high photoluminescence quantum yield. The X-ray imaging screens fabricated using TADF-Br chromophores exhibited highly improved X-ray sensitivity and imaging resolution compared with the TADF-H counterpart. More importantly, the high X-ray imaging resolution of >18.0 line pairs per millimetre achieved from the TADF-Br screen exceeds most reported organic and conventional inorganic scintillators. This study could help revive research on organic X-ray imaging scintillators and pave the way towards exciting applications for radiology and security screening. Heavy atoms like Cl, Br and I introduced into thermally activated delayed fluorescence chromophores can increase the X-ray absorption cross-section. Light yield of ~20,000 photons MeV–1, detection limit of 45.5 nGy s−1 and imaging resolution of >18.0 line pairs per millimetre is demonstrated.

Ultrafast Evaluation of Two-Photon Absorption with Simplified Time-Dependent Density Functional Theory.

This work presents the theoretical background to evaluate two-photon absorption (2PA) cross-sections in the framework of simplified time-dependent density functional theory (sTD-DFT). Our new implementation allows the ultrafast evaluation of 2PA cross-sections for large molecules based on a regular DFT ground-state determinant as well as a variant employing our tight-binding sTD-DFT-xTX flavor for very large systems. The method is benchmarked against higher-level calculations for trans-stilbene and typical fluorescent protein chromophores. For eGFP, a quadrupolar chromophore and its branched version, the flavine mono-nucleotide, and the iLOV protein, we compare sTD-DFT 2PA spectra to experimental ones. This includes extension and testing of our all-atom quantum chemistry methodology for the evaluation of 2PA for a system of ∼2000 atoms, providing striking agreement with the experimental spectrum.

Capsulation of red emission chromophore into the CoZn ZIF as nanozymes for on-site visual cascade detection of phosphate ions, o-phenylenediamine, and benzaldehyde

Accurate on-site profiling of the pollutants is of vital significance for estimating environmental pollution. Herein, we propose a paper-based fluorescence-sensing system to precisely report the level of multiple pollutants. A high-performance fluorescence-sensor for apparatus-free and visual on-site tandem precisely reporting phosphate ions (Pi), o-phenylenediamine (OPD), and benzaldehyde (BA) levels have been fabricated successfully by introducing synthesized red emission (>600 nm) fluorescent chromophore 10-(diethylamino)-3-hydroxy-5,6-dihydrobenzo [c]xanthen-12-ium (HTD) into the environment of CoZn zeolitic imidazolate framework (CoZn ZIF) by a simple stirring method. CoZn ZIF@HTD with the bimetallic nodes not merely provided main Zn2+ sites for specific recognition of Pi to generate an enhanced red fluorescent optical signal, Co3+/Co2+ exhibited excellent peroxidase-like activity for the catalytic oxidation of OPD substrate in the presence of H2O2 resulting in color changing from red to yellow. Subsequently, the obvious yellow fading of the OPDox species took place with the addition of BA. By virtue of the sensitively visual tandem detection of Pi, OPD, and BA, the sensor can be applied to real wastewater samples. Meanwhile, this fluorescent sensor was further adopted for practical application in confocal cell imaging and security inks. Overall, this work established a fluorescent sensing system with integrated multifunctional applications for environmental and biological samples, implying the great potential for simultaneous real-time cascade detection of various important pollutants with the merit of low-cost, time-saving, and easy-to-use.

Rational Design of a Fluorescent Chromophore as a Calcium Receptor via DFT and Multivariate Approaches.

Computational and experimental approaches were adopted to utilize a chromophore diglycolic functionalized fluorescein derivative as a Ca2+ receptor. Fluorescein diglycolic acid (Fl-DGA, 1) was synthesized and used in multivariate determination of Ca2+ and K+. Full-structure computation shows that the complexes of 1 and Ca2+ have comparable energies regardless of additional interaction with lactone moiety. The initial formation of diglycolic-Ca2+ complex followed by macrocyclization is thermodynamically disfavored. A U-shaped pre-organized 1 allows Ca2+ to interact simultaneously with diglycolic and lactone motifs. Both motifs actively participate in Ca2+ recognition and the eleven methylene units in the undecyl arm provides excellent flexibility for reorganization and optimum interaction. Principal component analysis (PCA) of computational molecular properties reveals a simple method in evaluating motifs for cation recognition. Fragment models support full-structure results that negative charge causes significant structural changes, but do not reproduce the full extent of C-O bond breaking observed in the latter. Experimental optical responses show that 1 is selective towards Ca2+ and discriminates against K+ and Mg2+. PCA of emission intensities affords distinct clusters of 0.01, 0.1 and 1 mM Ca2+ and K+, and suggests applicability of this technique for simultaneous determination of cationic plant macronutrients in precision agriculture and a wide variety of other applications.

Benchmarking time-dependent density functional theory for singlet excited states of thermally activated delayed fluorescence chromophores

Thermally activated delayed fluorescence (TADF) is the internal conversion of triplet excitons into singlet excitons via reverse intersystem crossing. TADF can significantly enhance the efficiency of organic light-emitting diodes (OLEDs). In order for a chromophore to display TADF the energy difference between its lowest singlet and lowest triplet states, ${S}_{1}$ and ${T}_{1}$, should be as small as possible. This requirement is facilitated by spatial separation between the frontier orbitals. Computer simulations based on time-dependent density functional theory (TDDFT) have been used extensively to predict the excited state properties of TADF chromophores. However, the accuracy of TDDFT largely depends on the choice of exchange-correlation functional. Here, we present a benchmark study of the performance of TDDFT based on different classes of hybrid functionals for 16 TADF chromophores consisting of different donor and acceptor moieties. We find that only the range-separated double hybrid functionals, $\ensuremath{\omega}\mathrm{B}2\mathrm{PLYP}$ and $\ensuremath{\omega}\mathrm{B}2\mathrm{GP}\text{\ensuremath{-}}\mathrm{PLYP}$, provide qualitatively correct predictions of the relative singlet excitation energies of different molecules, the spectral composition of excited states, and the energy ordering of intramolecular charge-transfer versus valence excited states. Therefore, we recommend using these functionals to assess prospective TADF chromophores. Nevertheless, further development is needed to improve the quantitative performance of TDDFT. These findings are important for our ability to computationally screen and design candidate TADF chromophores and advance the development of highly efficient OLEDs.

Construction and in-situ thermodynamics/kinetics studies on Ag-bridged g-C3N4-{0 0 2}/BiOBr-{0 0 1} facet Z–scheme heterojunction with crystal plane synergistic effect based on photocalorimetry - spectroscopy technology

In this study, a novel Ag-bridged g-C3N4-{002}/BiOBr-{001} facet Z–scheme heterojunction nanocomposites with synergistic effect of crystal plane had been created, and its photocatalytic degradation performance for Rhodamine 6G (Rh-6G) had been evaluated.The resulting g-C3N4@Ag@BiOBr not only followed the Z-scheme interface transfer mechanism, but also showed good synergy between the highly exposed g-C3N4-{002} and BiOBr-{001} crystal planes, exhibiting better Rh-6G degradation performance, compared with the two-component g-C3N4@Ag and g-C3N4@BiOBr heterojunction systems. Photocalorimetry-spectroscopic study on the thermodynamics/kinetics mechanism of g-C3N4@Ag@BiOBr photocatalytic degradation of Rh-6G helped to unlock the degradation mechanism. Three main heat exchanges were detected during the photocatalytic action, corresponding to the electron-hole pair generation, Rh-6G degradation by active species, and the final stable exothermic stage with a stable exothermic rate of (-0.4592 ± 0.2368) mJ·s−1. The whole process corresponded to that N-de-ethylated, deaminization and other cleavage of the fluorescent chromophore reaction for Rh-6G occurred rapidly within 30 min, then the mineralization and degradation of non-fluorescent chromophore intermediates which were the rate-determining steps eventually dominated, in accordance with the final stable exothermic stage of a pseudo-zero-order process. The results demonstrated that the photocatalytic degradation of Rh-6G over g-C3N4@Ag@BiOBr was a pseudo-zero-order reaction rather than a first-order reaction as measured by universal spectroscopic method. This study not only created highly efficient composites photocatalysts, but also shed new light on revealing the photocatalytic mechanism using the complete expression of thermodynamics, kinetics and spectral kinetics.

Action-Absorption Spectroscopy at the Band Origin of the Deprotonated Green Fluorescent Protein Chromophore In Vacuo.

The application of action spectroscopy in connection with determination of the S0 to S1 band origin in the GFP anion model chromophore (deprotonated HBDI) is discussed. We specifically address the consequences of the lowest vibrational levels in S1 being located behind a potential-energy barrier that inhibits internal conversion to the S0 electronic ground state. Action spectroscopy based on consecutive absorption of two photons together with internal conversion will as a consequence reveal an apparent band origin that is significantly blue-shifted.

Synthesis and Anticholinesterase Activity of Some 2,5-Disubstituted Imidazol-4-ones Derived from the Green Fluorescent Protein Chromophore

Synthesis and Anticholinesterase Activity of Some 2,5-Disubstituted Imidazol-4-ones Derived from the Green Fluorescent Protein Chromophore

Near-infrared photosensitive dyes based on red fluorescence protein chromophore analogue for photodynamic therapy and two-photon fluorescence imaging

In this paper, two photosensitizers RFP-OH and RFP-Ac based on red fluorescent protein chromophore analogue with NIR emission was synthesized for photodynamic therapy (PDT) in A-549 cells and zebrafish. After the introduction of p-hydroxystyrene and acryloyl ester, the emission wavelengths of RFP-OH and RFP-Ac were red-shifted to the NIR band, which were 721 nm and 758 nm, respectively. And the stokes shift of RFP-OH and RFP-Ac was as large as 228 nm and 260 nm, respectively. The singlet oxygen yield of RFP-OH and RFP-Ac was 35.6% and 28.8%, respectively. Tumor cells are rich in biological thiols, such as Cys. After RFP-Ac encountered with Cys, the singlet oxygen yield of the products increased to 34.1%. In addition, DFT calculations showed that RFP-OH can transition to triplet state through intersystem crossing after being excited to singlet state, thereby enhancing the singlet oxygen yield. Both RFP-OH and RFP-Ac showed low dark toxicity (≥91.02%), high photo toxicity, and good PDT effects in A-549 cells and zebrafish. More importantly, RFP-OH exhibited superior two-photon fluorescence imaging properties in zebrafish. In a word, considering to the positive results of the experiments, RFP-OH and RFP-Ac had a great potential for PDT in anti-cancer therapy.

SfGFP throws light on the early stages of β-barrel amyloidogenesis

The accumulation of β-sheet-rich protein aggregates, amyloid fibrils, accompanies severe pathologies (Alzheimer's, Parkinson's diseases, ALS, etc.). The high amyloidogenicity of proteins with a native β-barrel structure, and the amyloidogenic peptides ability to form a universal cylindrin-like oligomeric state were proven. The mechanisms for the proteins' transformation from this state to a fibrillar one are still not fully understood. We defined the structural rearrangements of the amyloidogenic β-barrel superfolder GFP (sfGFP) prior to fibrillogenesis using its tryptophan and chromophore fluorescence. We characterized the early intermediate “native-like” state preserving the integrity of the sfGFP β-barrel scaffold despite the partial distortion of the three β-strands closing it. The interaction between the “melted” regions of the protein leads to the assembly of high molecular weight complexes, which are not dynamic structures but are less stable and less cytotoxic than mature amyloids. Additional contacts of sfGFP monomers facilitate the global reorganization of its structure and stabilization of the second intermediate state in which the β-barrel opens and some of the native α-helices and disordered regions refold into non-native β-strands, which, along with native β-strands, form an amyloid fiber. Reported sfGFP structural transformations may occur during the fibrillogenesis of other β-barrel proteins, and the identified intermediate states are likely universal. Thus sfGFP can be used as a sensing platform to develop therapeutic agents inhibiting amyloidogenesis through interaction with protein intermediates and destroying low-stable aggregates formed at the early stages of fibrillogenesis.

Biocompatible Abscisic Acid-Sensing Supramolecular Hybridization Probe for Spatiotemporal Fluorescence Imaging in Plant Tissues.

Achieving detection of the phytohormone abscisic acid (ABA) is of critical importance for understanding plant growth and development. We report a hybrid supramolecular fluorescent probe that uses bovine serum albumin (BSA) as a host. Aggregation-induced emission of fluorescent chromophores (AIEgens) enables luminescence in the presence of BSA. ABA and its aptamer act as a switch to trigger this fluorescent system, the strategy that exhibits high sensitivity to abscisic acid with a detection limit of 0.098 nM. The probe test strip also enables visualization of ABA content from plants by colorimetric observation with the naked eye. In particular, the high biocompatibility and small molecular size of the prepared fluorescent probe allow for effective monitoring of ABA in plant tissues by fluorescence imaging. This strategy provides a new perspective to achieve the detection of endogenous and exogenous ABA in plants and has important implications for plant biology research.

Multi-Decker Emissive Supramolecular Architectures Based on Shape-Complementary Ligands Pair.

Dye aggregates have attracted a great deal of attention due to their widespread applications in organic light-emitting devices, light-harvesting systems, etc. However, the strategies to precisely control chromophores with specific spatial arrangements still remain a great challenge. In this work, a series of double- and triple-decker supramolecular complexes are successfully constructed by coordination-driven self-assembly of carefully designed shape-complementary ligands, one claw-like tetraphenylethylene (TPE)-based host ligand and three tetratopic or ditopic guest ligands. The spatial configurations of these assemblies (one double-decker and three "S-shaped" or "X-shaped" triple-decker structures) depend on the angles of these TPE-derived ligands. Notably, the three triple-decker structures are geometric isomers. Furthermore, photophysical studies show that these complexes exhibit different ratios of radiative (kr ) and non-radiative (knr ) rate constant due to the different spatial arrangements of TPE moieties. This study provides not only a unique strategy for the construction of multi-stacks with specific spatial arrangement, but also a promising platform for investigating the aggregation behavior of fluorescent chromophores.

Highly Fluorescent Bipyrrole-Based Tetra-BF2 Flag-Hinge Chromophores: Achieving Multicolor and Circularly Polarized Luminescence.

This study reports the facile syntheses of tetra-boron difluoride (tetra-BF2 ) complexes, flag-hinge-like molecules that exhibit intense green-to-orange luminescence in solution and yellow-to-red emission in the solid states. Single-crystal structure analysis and density functional theory calculations suggested a bent structure of this series of compounds. The complexes also exhibited excellent optical properties, with quantum yields reaching 100 % and a large Stokes shift. These properties were attributed to the altered bending angle of the molecule in the S1 excited state. As the rotational motion was suppressed around the 2,2'-bipyrrole axis, atropisomers with axial chirality were formed, which are optically resolvable into (R) and (S)-enantiomers through a chiral column. The atropisomers thus function as circularly polarized luminescent (CPL) materials, in which the color (green, green-yellow, and yellow) can be varied by controlling the aggregation state. This rational design of multi-BF2 complexes can potentially realize novel photofunctional materials.

"On/Off" Switchable Sequential Light-Harvesting Systems Based on Controllable Protein Nanosheets for Regulation of Photocatalysis.

A controllable protein nanostructures-based "On/Off" switchable artificial light-harvesting system (LHS) with sequential multistep energy transfer and photocatalysis was reported herein for mimicking the natural LHS in both structure and function. Single-layered protein nanosheets were first constructed via a reversible covalent self-assembly strategy using cricoid stable protein one (SP1) as building blocks to realize an ordered arrangement of pigments. Fluorescent chromophores like carbon dots (CDs) can be precisely distributed on the protein nanosheets superficially via electrostatic interactions and make the ratio between donors and acceptors adjustable. After being anchored with a photocatalysis center (eosin-5-isothiocyanate, EY), the constructed LHS could sequentially transfer energy between two kinds of chromophores (CD1 and CD2), and further transfer to EY center with a high efficiency of 84%. Interestingly, the Förster resonance energy transfer (FRET) process of our LHS could be reversibly "On/Off" switched by the redox regulated assembly and disassembly of SP1 building blocks. Moreover, the LHS has been further proved to promote the yield of a model cross-coupling hydrogen evolution reaction and regulate the process of the reaction with the FRET process "On/Off" state.

TDDFT Investigation of the Raman and Resonant Raman Spectra of Fluorescent Protein Chromophore Models.

The off-resonance and resonant Raman spectra have been simulated for models of fluorescent protein chromophores, those of the green fluorescent protein (GFP, called FP1) and of DsRed (called FP2), which presents a longer π-conjugated path, with the aim of providing a systematic investigation of structural but also computational aspects. These were performed at the (time-dependent) density functional theory [(TD)DFT] level. The off-resonance intensities have been calculated from the derivatives of the frequency-dependent polarizability with respect to the normal coordinates while the resonant ones have been evaluated using Huang-Rhys factors determined from the gradients of the excitation energies with respect to the normal coordinates. When applied with the M05 meta-GGA exchange-correlation functional, this simple computational scheme can reproduce most of the experimental Raman signatures of FP1 in its protonated and deprotonated forms, the differences of vibrational signatures of the cis (Z) and trans (E) isomers, as well as their changes as a function of the excitation wavelength. On the other hand, testing the predictions made for FP2 would require new experimental work. It was also observed that simulations with methods that inadequately predict the resonant Raman spectra could nevertheless produce a UV-vis absorption spectrum that is quite similar to the one obtained with better methods, once realistic peak broadening has been applied.

S-Tetrazine-Bridged Photochromic Aromatic Framework Material.

Integrating fluorescent chromophores in aromatic frameworks could not only prevent aggregation-induced quenching caused by the π–π stacking interaction between the chromophore components but also confer new fluorescence properties. Herein, we report the fabrication of s-tetrazine-bridged aromatic frameworks TzAF by the incorporation of the smallest aromatic fluorophore, s-tetrazine (Tz), into the skeleton of a tetrahedrally connected lattice of aromatic frameworks. The thin films of TzAF coated on silica gel plates were found to exhibit reversible photoswitching fluorescence characteristics under alternate UV and visible-light irradiations with excellent fluorescence stability and high on/off contrast. The repeatable “on/off”fluorescence photoswitchability of the TzAF thin films was mechanistically attributed to light-induced reversible transformation between TzAF’s neutral and radical states.

Fluorescence Switching for Temperature Sensing in Water.

A water-soluble thermochromic molecular switch with spectrally resolved fluorescence in its two interconvertible states can be assembled in three synthetic steps by integrating a fluorescent coumarin chromophore, a hydrophilic oligo(ethylene glycol) chain, and a switchable oxazole heterocycle in the same covalent skeleton. Measurements of its two emissions in separate detection channels of a fluorescence microscope permit the noninvasive and ratiometric sensing of temperature at the micrometer level with millisecond response in aqueous solutions and within hydrogel matrices. The ratiometric optical output of this fluorescent molecular switch overcomes the limitations of single-wavelength fluorescent probes and enables noninvasive temperature mapping at length scales that are not accessible to conventional thermometers based on physical contact.

Principles, modulation, and applications of fluorescent protein chromophores

Fluorescent proteins (FPs) have gained much attention over the last few decades as powerful tools in bioimaging since the discovery of green fluorescent protein (GFP) in the 1960s. The mechanism of FP bioluminenscence has been well-studied, and new variants with improved photophysical properties are being constantly generated. In this review, a brief history of GFP along with its biogenesis is first provided. Next, the fluorescent and quenching mechanism governing the photophysical property of GFP is elaborated. Most importantly, we seek to introduce the expanding family of FP derivatives that mimics the chromophore core structure of FPs. Multiple physical and chemical strategies have been discussed to minimize the inherent fluorescence quenching effect of FP derivatives. Finally, we briefly overview the biological application of FP derivatives, with a focus on fluorescent RNA aptamer and recently reported protein aggregation detection probes. Through citing and discussing the most important works in this field, this review aims to provide a general photophysical understanding regarding the luminescence phenomenon of GFP and its derivatives, as well as chemical strategies to design functional FP derivatives.